红豆杉细胞合成紫杉醇的分子调控机理
|
摘要
植物材料在离体培养过程中常发生遗传变异。这些变异包括外部形态、育性、生长势、抗性和次生代谢等各个方面。植物细胞在离体培养过程中发生的遗传变异往往导致细胞在大规模培养过程中生产次生代谢产物的含量不稳定可控。具体表现为：第一，在不同的培养时期和培养环境下，细胞表观形态、生长速率和合成次生代谢产物的能力发生变化；第二，在相同的传代次数和培养条件下，不同小细胞团的形态颜色发生变化、生长速率不一致、细胞有丝分裂周期不同步、合成次生代谢产物能力发生变化。这些现象导致科学家们筛选得到的高产优质的种质细胞资源无法获得长期有效的应用，影响植物细胞大规模培养生产次生代谢产物的工业化进程。因此，研究植物细胞在离体培养过程中发生遗传变异的影响因素，探讨变异发生的遗传基础，揭示次生代谢产物在细胞离体培养中发生变化的分子调控机理是解决植物细胞工业化应用的前提和基础。本文以离体培养的红豆杉细胞为研究对象，从细胞学、紫杉醇合成关键酶基因的表达谱、基因组DNA遗传和表观遗传学等层面系统研究红豆杉细胞在离体培养过程中遗传变异规律及其分子机理和影响因素，揭示红豆杉细胞合成紫杉醇的分子调控机理，为红豆杉细胞大规模培养生产紫杉醇奠定基础，取得了以下结果：
建立了红豆杉细胞遗传变异分析方法和技术体系，如实时荧光定量PCR技术检测紫杉醇合成关键酶基因表达量、AFLP和MSAP分子标记技术检测红豆杉细胞遗传和表观遗传变异、HPLC技术检测红豆杉细胞DNA甲基化水平。
离体培养过程中红豆杉细胞遗传变异的分子机理：随着离体培养时间延长，细胞褐化减轻、生长率增加、细胞出现多核和不对称分裂现象、染色体出现四倍体和非整倍体变异、紫杉醇和紫杉烷类含量下降、紫杉醇合成关键酶基因的表达量下降；AFLP分析表明基因组DNA序列的稳定性较好，未出现显著变异，HPLC和MSAP分析显示DNA甲基化水平和模式在红豆杉细胞长期离体培养过程中发生改变，基因组DNA的甲基化修饰水平逐渐升高，且紫杉醇生物合成与DNA甲基化水平密切相关。
环境因子影响红豆杉细胞紫杉醇含量的分子机理：改变固液培养方式、光照条件、添加条件培养液等措施仅能在短期内改变紫杉醇合成能力。调整培养基激素浓度能长期影响红豆杉细胞中紫杉醇的合成。在高激素浓度培养基上长期离体培养的红豆杉细胞紫杉醇含量下降、细胞小不均一，形态不规则，出现巨型细胞、空泡细胞和小细胞、紫杉醇合成关键酶基因的表达量下降。基因组DNA序列比较稳定，但是出现了DNA甲基化水平和模式的显著变化。在低激素浓度培养基上长期离体培养的红豆杉细胞的遗传变异现象与之相反。说明植物激素作用可能是影响红豆杉细胞长期离体培养过程中发生遗传变异的因素之一，且与高激素条件下引起的细胞中DNA甲基化水平变异密切相关。
低温缓慢生长法培养红豆杉细胞可以减少红豆杉细胞在长期离体培养过程中的传代次数，但在恢复培养过程中仍发生了遗传变异：红豆杉细胞分别在低温保存30 d、60 d、180 d和1 a后恢复培养30 d、60 d、180 d过程中，随着紫杉醇和紫杉烷含量的改变，紫杉醇合成关键酶基因的表达谱、基因组DNA序列稳定性、DNA甲基化水平和模式均发生了相应的改变，其中，DNA甲基化水平的变异与紫杉醇含量的改变密切相关。
上述研究结果均证明紫杉醇含量与DNA甲基化水平始终密切相关。外源添加5-Aza-CdR，能抑制红豆杉DNA甲基转移酶(CMT)的活性，降低红豆杉DNA甲基化水平，提高紫杉醇合成关键酶基因的表达，从而提高细胞合成紫杉醇的能力。
在相同的传代次数和培养条件下，同步化处理能有效控制部分小细胞团引起的紫杉醇合成能力发生变化的现象。研究表明未经低温同步化的红豆杉细胞中的关键酶基因DXR、HMGR、GGPPS和DBAT的表达量在MJ诱导24 h后均迅速下降，但在低温同步化的细胞中DBAT基因表达量增加，4个关键基因的表达量下降减缓，诱导60h后仍维持较高水平。说明DBAT基因表达量增加以及一系列其他紫杉醇合成关键酶基因在转录水平的高效持续表达是同步化稳定提高紫杉醇产量的主要分子机理之一。
红豆杉细胞在离体培养过程中，紫杉醇生物合成能力受到合成紫杉醇关键酶基因转录水平的调控。DNA甲基化／去甲基化模式可能是启动关键酶基因转录的“开关”，从而表现为DNA甲基化水平与与紫杉醇合成能力的密切相关。因此，DNA甲基化引起的紫杉醇合成关键酶基因表达量的改变可能是红豆杉细胞离体培养过程中调控紫杉醇合成的重要分子机理之一。
In vitro culture of plant materials often led to genetic variation,described assomaclonal variation,including morphology,fertility,growth potentiality,resistibility andsecondary metabolism aspects.Genetic variation can result in unstable and uncontrollablesecondary metabolite production in large-scale plant cell culture:firstly,in differentgrowing periods and culture environments,cell morphology,growth rate and ability ofsecondary metabolite production have changed;secondly,in the same passage times andculture environments,different small calli demonstrated as different morphology,color,growth rate,asynchronization of cell mitosis cycle and ability of secondary metaboliteproduction.For these reasons,the high-yield germplasm cell can not be applied insecondary metabolite production by large-scale plant cell culture efficiently.Thus,study onimpacting factors and molecular mechanism of genetic variation during the process of invitro plant cell culture is the foundation of plant cell industrial application.In this study,wetake Taxus media cv.Hicksii as research object and try to discover molecular regulationmechanism of taxol biosynthesis in Taxus media cv.Hicksii cell line at chromatosome,genetranscript level,genetic level and epigenetic level.Detailed study as followed:
Genetic variation of Taxus during long-term in vitro taxus cell culture:Gradual loss ofpaclitaxel yield in taxus cell line during 5 years in vitro cell culture was observed,withdecreasing cell browning,increasing growth rate and chromatosome variation.Transcriptlevel of four genes encoding key enzymes in the paclitaxel biosynthesis pathway wereheavily lower and little DNA sequence polymorphism were found by AFLP assayed.While,both HPLC and MSAP methods displayed variation of DNA methylation pattern ofdifferent samples.
Effect of environment on genetic stability of Taxus media cv.Hicksii cell duringlong-term in vitro taxus cell culture:compared with environment factors,as culture method,light condition,condition medium,plant auxin play an important role in taxol contentvariation.Auxin content impacted not only taxol production,but also cell phenotype,transcript level of four genes encoding key enzymes in the paclitaxel biosynthesis pathway,DNA methylation level and pattern.
Taxus cell line genetic stability during process of low temperature preservation:taxus cell line was cultured in low temperature for 30 d,60 d,180 d and 1 a,and recovered tonormal temperature for 30 d,60 d,180 d,separately.Along with taxol and 10-DAB contentvariation,cell phenotype,transcript level of four genes encoding key enzymes in thepaclitaxel biosynthesis pathway,DNA methylation level and pattern happened variation.Repeated low temperature preservation could maintain taxus cell line genetic stability inlong-term.
Correlation analysis between taxol content of Taxus media cv.Hicksii and cellbiomass,key enzyme expression,DNA methylation suggested that DNA methylationshowed a significant correlation with taxol content of Taxus media cv.Hicksii.Taxus cellline treated with 5-Aza-CdR could inhibit CMT activity and lower DNA methylationlevel.De-methylation progress maybe stimulate the expression of serial genes related withtaxol biosynthesis.Therefore,DNA methylation maybe regulate taxol production in Taxuscell line.
In addition,molecular regulation mechanism of cold synchronization treatment wasstudied.The expression levels of DXR,HMGR,GGPPS and DBAT decreased rapidly after24 h induced by MJ in cells untreated with cold.On the contrary,expression level of DBATwas improved and expression level of those genes decreased slowly in cells with coldtreatment,and the genes expression maintained higher levels after 60 h induced by MJ.Incells with cold synchronization treatment and MJ induction,the high and steady expressionof taxol biosynthetic pathway key genes maybe one reason for the steady improvement oftaxol content.
建立了红豆杉细胞遗传变异分析方法和技术体系，如实时荧光定量PCR技术检测紫杉醇合成关键酶基因表达量、AFLP和MSAP分子标记技术检测红豆杉细胞遗传和表观遗传变异、HPLC技术检测红豆杉细胞DNA甲基化水平。
离体培养过程中红豆杉细胞遗传变异的分子机理：随着离体培养时间延长，细胞褐化减轻、生长率增加、细胞出现多核和不对称分裂现象、染色体出现四倍体和非整倍体变异、紫杉醇和紫杉烷类含量下降、紫杉醇合成关键酶基因的表达量下降；AFLP分析表明基因组DNA序列的稳定性较好，未出现显著变异，HPLC和MSAP分析显示DNA甲基化水平和模式在红豆杉细胞长期离体培养过程中发生改变，基因组DNA的甲基化修饰水平逐渐升高，且紫杉醇生物合成与DNA甲基化水平密切相关。
环境因子影响红豆杉细胞紫杉醇含量的分子机理：改变固液培养方式、光照条件、添加条件培养液等措施仅能在短期内改变紫杉醇合成能力。调整培养基激素浓度能长期影响红豆杉细胞中紫杉醇的合成。在高激素浓度培养基上长期离体培养的红豆杉细胞紫杉醇含量下降、细胞小不均一，形态不规则，出现巨型细胞、空泡细胞和小细胞、紫杉醇合成关键酶基因的表达量下降。基因组DNA序列比较稳定，但是出现了DNA甲基化水平和模式的显著变化。在低激素浓度培养基上长期离体培养的红豆杉细胞的遗传变异现象与之相反。说明植物激素作用可能是影响红豆杉细胞长期离体培养过程中发生遗传变异的因素之一，且与高激素条件下引起的细胞中DNA甲基化水平变异密切相关。
低温缓慢生长法培养红豆杉细胞可以减少红豆杉细胞在长期离体培养过程中的传代次数，但在恢复培养过程中仍发生了遗传变异：红豆杉细胞分别在低温保存30 d、60 d、180 d和1 a后恢复培养30 d、60 d、180 d过程中，随着紫杉醇和紫杉烷含量的改变，紫杉醇合成关键酶基因的表达谱、基因组DNA序列稳定性、DNA甲基化水平和模式均发生了相应的改变，其中，DNA甲基化水平的变异与紫杉醇含量的改变密切相关。
上述研究结果均证明紫杉醇含量与DNA甲基化水平始终密切相关。外源添加5-Aza-CdR，能抑制红豆杉DNA甲基转移酶(CMT)的活性，降低红豆杉DNA甲基化水平，提高紫杉醇合成关键酶基因的表达，从而提高细胞合成紫杉醇的能力。
在相同的传代次数和培养条件下，同步化处理能有效控制部分小细胞团引起的紫杉醇合成能力发生变化的现象。研究表明未经低温同步化的红豆杉细胞中的关键酶基因DXR、HMGR、GGPPS和DBAT的表达量在MJ诱导24 h后均迅速下降，但在低温同步化的细胞中DBAT基因表达量增加，4个关键基因的表达量下降减缓，诱导60h后仍维持较高水平。说明DBAT基因表达量增加以及一系列其他紫杉醇合成关键酶基因在转录水平的高效持续表达是同步化稳定提高紫杉醇产量的主要分子机理之一。
红豆杉细胞在离体培养过程中，紫杉醇生物合成能力受到合成紫杉醇关键酶基因转录水平的调控。DNA甲基化／去甲基化模式可能是启动关键酶基因转录的“开关”，从而表现为DNA甲基化水平与与紫杉醇合成能力的密切相关。因此，DNA甲基化引起的紫杉醇合成关键酶基因表达量的改变可能是红豆杉细胞离体培养过程中调控紫杉醇合成的重要分子机理之一。
In vitro culture of plant materials often led to genetic variation,described assomaclonal variation,including morphology,fertility,growth potentiality,resistibility andsecondary metabolism aspects.Genetic variation can result in unstable and uncontrollablesecondary metabolite production in large-scale plant cell culture:firstly,in differentgrowing periods and culture environments,cell morphology,growth rate and ability ofsecondary metabolite production have changed;secondly,in the same passage times andculture environments,different small calli demonstrated as different morphology,color,growth rate,asynchronization of cell mitosis cycle and ability of secondary metaboliteproduction.For these reasons,the high-yield germplasm cell can not be applied insecondary metabolite production by large-scale plant cell culture efficiently.Thus,study onimpacting factors and molecular mechanism of genetic variation during the process of invitro plant cell culture is the foundation of plant cell industrial application.In this study,wetake Taxus media cv.Hicksii as research object and try to discover molecular regulationmechanism of taxol biosynthesis in Taxus media cv.Hicksii cell line at chromatosome,genetranscript level,genetic level and epigenetic level.Detailed study as followed:
Genetic variation of Taxus during long-term in vitro taxus cell culture:Gradual loss ofpaclitaxel yield in taxus cell line during 5 years in vitro cell culture was observed,withdecreasing cell browning,increasing growth rate and chromatosome variation.Transcriptlevel of four genes encoding key enzymes in the paclitaxel biosynthesis pathway wereheavily lower and little DNA sequence polymorphism were found by AFLP assayed.While,both HPLC and MSAP methods displayed variation of DNA methylation pattern ofdifferent samples.
Effect of environment on genetic stability of Taxus media cv.Hicksii cell duringlong-term in vitro taxus cell culture:compared with environment factors,as culture method,light condition,condition medium,plant auxin play an important role in taxol contentvariation.Auxin content impacted not only taxol production,but also cell phenotype,transcript level of four genes encoding key enzymes in the paclitaxel biosynthesis pathway,DNA methylation level and pattern.
Taxus cell line genetic stability during process of low temperature preservation:taxus cell line was cultured in low temperature for 30 d,60 d,180 d and 1 a,and recovered tonormal temperature for 30 d,60 d,180 d,separately.Along with taxol and 10-DAB contentvariation,cell phenotype,transcript level of four genes encoding key enzymes in thepaclitaxel biosynthesis pathway,DNA methylation level and pattern happened variation.Repeated low temperature preservation could maintain taxus cell line genetic stability inlong-term.
Correlation analysis between taxol content of Taxus media cv.Hicksii and cellbiomass,key enzyme expression,DNA methylation suggested that DNA methylationshowed a significant correlation with taxol content of Taxus media cv.Hicksii.Taxus cellline treated with 5-Aza-CdR could inhibit CMT activity and lower DNA methylationlevel.De-methylation progress maybe stimulate the expression of serial genes related withtaxol biosynthesis.Therefore,DNA methylation maybe regulate taxol production in Taxuscell line.
In addition,molecular regulation mechanism of cold synchronization treatment wasstudied.The expression levels of DXR,HMGR,GGPPS and DBAT decreased rapidly after24 h induced by MJ in cells untreated with cold.On the contrary,expression level of DBATwas improved and expression level of those genes decreased slowly in cells with coldtreatment,and the genes expression maintained higher levels after 60 h induced by MJ.Incells with cold synchronization treatment and MJ induction,the high and steady expressionof taxol biosynthetic pathway key genes maybe one reason for the steady improvement oftaxol content.
引文
[1]Wani M C,Taylor H L,Wall M E.Plant antitumor agents.Ⅵ,the isolation and structure of taxol,a novel antileukemic and antitumor agent from taxus brevifolia.J Am Chem Soc,1971,27:433～437.
[2]Dong H D,Zhong J J.Enhanced taxane productivity in bioreactor cultivation of taxus chinensis cells by combining elicitation,sucrose feeding and ethylene incorporation.Enzyme Microb Tech,2002,31:116～121.
[3]Wang Y D,Yuan Y J,Wu J C.Induction studies of methyl jasmonate and salicylic acid on taxane production in suspension cultures of taxus chinensis var.mairei.Biochem Engin J,2004,19:259～326
[4]Croteau R,Ketchum R E B,Long R Met al.Taxol biosynthesis and molecular genetics.Phytochem Reviews,2006,5:75.
[5]Ketchum R E,Gibson D M,Croteau R Bet al.The kinetics of taxoid accumulation in cell suspension cultures of taxus following elicitation with methyl jasmonate.Biotechnol Bioeng,1999,62:97～105.
[6]Yukimune Y,Tabata H,Higashi Yet al.Methyl jasmonate-induced overproduction of paclitaxel and baccatin Ⅲ in taxus cell suspension cultures.Nat.Bioteehnol,1996,14:1129～1132.
[7]Pyo S,Parka H,Song B et al.A large-scale purification of paclitaxel from cell cultures of taxus chinensis,2004,39:1985～1991.
[8]张振学，张海霞，杨庆林．紫杉醇构效关系及其类似物的研究开发进展．天然产物研究与开发，2005，17：810～817．
[9]Zhang W,Curtin C,Franco C.Towards manipulation of post-biosynthetic events in secondary metabolism of plant cell cultures.Enzyme Microb Tech,2002,30:688～696.
[10]Vanisree M,Lee C Y,Lo S F et al.Studies on the production of some important secondary metabolites from medicinal plants by plant tissue cultures.Bot Bull Acad Sin,2004,45:1～22.
[11] Liao Z H, Chen M, Gong Y F et al. A new farnesyl diphosphate synthase gene from taxus media rehder. cloning, characterization and functional complementation. J Iintegr Plant Biol, 2006,48: 692-699.
[12] Jennewein S, Park H,Dejong J Met al. Coexpression in yeast of taxus cytochrome p450 reductase with cytochrome p450 oxygenases involved in taxol biosynthesis. Biotechnol Bioeng, 2005, 89: 588-598.
[13] Walker K D, Klettke K, Akiyama T et al. Cloning, heterologous expression, and characterization of a phenylalanine aminomutase involved in taxol biosynthesis. J Biol Chem, 2004, 279: 53947-53954.
[14] Jennewein S, Long R M, Williams R Met al. Cytochrome p450 taxadiene 5a-hydroxylase, a mechanistically unusual monooxygenase catalyzing the first oxygenation step of taxol biosynthesis. Chem Biol, 2004,11: 379-387.
[15] Chau M D, Jennewein S, Walker K et al. Taxol biosynthesis: molecular cloning and characterization of a cytochrome p450 taxoid 7P-hydroxylase. Chem Biol, 2004, 11:663-672.
[16] Kirschberg T A, Vandeusen C L, Rothbard J B et al. Arginine-based molecular transporters the synthesis and chemical evaluation of releasable taxol-transporter conjugates. Org Lett, 2003, 5: 3459-3462.
[17] Walker K, Croteau R. Molecular cloning of a 10-deacetylbaccatinⅢ-10-o-acetyl transferase cDNA from taxus and functional expression in escherichia coli. Proc Natl Acad Sci USA, 2000,97: 583-587.
[18] Hefner J, Ketchum R E, Croteau R. Cloning and functional expression of a cdna encoding geranylgeranyl diphosphate synthase from taxus canadensis and assessment of the role of this prenyltransferase in cells induced for taxol production. Arch Biochem Biophys, 1998, 360: 62-74.
[19] Tr Ehin C, Ahn I, Perennes C et al. Cloning of upstream sequences responsible for cell cycle regulation of the nicotiana sylvestris cycbl gene. Plant Mol Biol, 1997, 35:667-672.
[20] Hefner J, Rubenstein S M, Ketchum R E et al. Cytochrome p450-catalyzed hydroxylation of taxa-4(5), 11(12)-diene to taxa-4(20), 11(12)-dien-5a-ol: the first oxygenation step in taxol biosynthesis.Chem Biol,1996,3:479～489.
[21]余龙江，蔡永君，兰文智．红豆杉细胞培养生产紫杉醇产量稳定性的探讨．广西植物，2002，22：85～88．
[22]赵春芳，余龙江，李丽琴等．氧化水解法提高红豆杉浸膏中紫杉醇产率的研究．天然产物研究与开发，2006，18：95～99．
[23]Kim B J,Gibson D M,Shuler M L.Effect of subculture and elicitation on instability of taxol production in taxus sp.suspension cultures.Biotechnol Prog,2004,20:1666～1673.
[24]Sierra M I,Van Der Heijden R,Van Der Leer T et al.Stability of alkaloid production in cell suspension cultures of tabernaemontana divaricata during long-term subculture.Plant Cell Tiss Org Cult,1992,28:59～68.
[25]Zhang C H,Fevereiro P S.The effect of heat shock on paclitaxel production in taxus yunnanensis cell suspension cultures:role of abscisic acid-pretreatment.Biotechnol Bioeng,2007,96:506～514.
[26]陈永勤，朱蔚华，吴蕴祺等．不同种类红豆杉愈伤组织的诱导及紫杉醇含量的差异．中草药，2000，31：216～218．
[27]杜亚填，陈建华，许建宇等．植物生长调节剂对南方红豆杉愈伤组织培养和紫杉醇合成的影响．天然产物研究与开发，2006，18：569～576．
[28]盛长忠，王淑芳，王勇等．pH对红豆杉愈伤组织生长、PAL活性和紫杉醇含量的影响．中草药，2001，32：929～931．
[29]Kim J H，Yun J H，Hwang Y S．Production of taxol and relied taxanes in taxus brevifolia call cultures：effect of sugar．Biotechnol Lett，1995，17：101～106．
[30]陈永勤，朱蔚华，吴蕴祺．组培条件对云南红豆杉愈伤组织生长和形成紫杉醇的影响．中国中药杂志，2000，25：269～272．
[31]王艳东，葛志强，马振毅等．外加紫杉醇后南方红豆杉细胞的耐受性及其凋亡的研究．高校化学工程学报，2002，16：421～425．
[32]Larkin P J,Scowcroft W R.Somaclonal variation:a novel source of variability from cell cultures for plant improvement.Theor Appl Genet,1981,60:197～214.
[33]Sun Z Y,Han L,Li Y F.Progress in the study and application of plant smaclonal variation.Acta Agriculture Nuleatae Sinica,2005,6:479～484.
[34]Peredo E L,Angeles Revilla M,Arroyo-Garcia R.Assessment of genetic and epigenetic variation in hop plants regenerated from sequential subcultures of organogenic calli.J Plant Physiol,2006,163:1071～1079.
[35]李晓玲，丛娟，于晓明等．植物体细胞无性系变异研究进展．植物学通报，2008，25：121～128．
[36]Amato F.Cytogenetics of plant cell and tissue culture and their regenerates.Crc Crit Rev Plant Sci,1985,8:72～73.
[37]Johnson S S,Phillips R L,Rines H W.Possible role ofheterochromatin chromosome breakage induced by tissue culture in oats(avena sativa 1.).Genome,1987,29:439～446.
[38]高东迎，郭士伟，李霞等．水稻体细胞无性系变异．植物学通报，2002，19：749～755．
[39]Noro Y,Takano-Shimizu T,Syono K.Genetic variations in rice in vitro cultures at the epsps-rps20 region.Theor Appl Genet,2007,114:705～711.
[40]Brettell R I S,Pallotta M A,Gustafson J P et al.Variation at the nor loci in triticale derived from tissue culture.Theor Appl Genet,1986,71:637～643.
[41]Hirochika H,Sugimoto K,Otsuki Y.Retrotransposons of rice involved in mutations induced by tissue culture.Proc Natl Acad Sci U S A,1996,93:7783～7788.
[42]Phillips R L,Keappler S M,Olhoft P.Genetic instability of plant tissue cultures:breakdown of normal controls.Proc Natl Acad Sci USA,1994,91:5222～5226.
[43]Ng H H,Bird A P.DNA methylation and chromatin modification.Curr Opin Genet Dev,1999,9:158～163.
[44]Curradi M,Izzo A,Badaracco Get al.Molecular mechanisms of gene silencing mediated by DNA methylation.Mol Cell Biol,2002,22:3157～3173.
[45]Long L K,Lin X Y,Zhai J Z et al.Heritable alteration in DNA methylation pattern occurred specifically at mobile elements in rice plants following hydrostatic pressurization.Biochem Biophys Res Commun,2006,340:369～376.
[46].Morrish F M,Hanna W W.The expression and perpetuation of inherent somaclonal variation in regenerants from embryogenic cultures ofpennisetum glaucum(1.)r.br.(pearl millet).Theor Appl Genet,1990,80:409～416.
[47]Peschke V M,Phillips R L.Genetic implication of somaclonal variation in plants.Adv Genet,1992,30:41～75.
[48]刁现民，段胜军，陈振玲等．谷子体细胞无性系变异分析．中国农业科学，1999，32：21～26．
[49]Deus-Neumann B,Zenk M H.Instability of indole alkaloid production in catharanthus roseus cell suspension cultures.Planta Medica,1984,50:427～431.
[50]Kim B J,Gibson D M,Shuler M L.Effect of subculture and elicitation on instability of taxol production in taxus sp.suspension cultures.Biotechnol Prog,2004,20:1666～1673.
[51]Dolezel J,Lucretti S,Novak F J.The influence of 2,4-dichlorophenoxyacetic acid on cell cycle kinetics and sister-chromatid exchange frequency in garlic(allium sativa 1.)meristem cells.Biol Plant,1987,29:253～257.
[52]Rubluoa A,Hernandezb T M,Duvalal K et al.Auxin induced morphogenetic responses in long-term in vitro subcultured mammillaria san-angelensis sanchez-mejorada(cactaceae).Scientia Horticulturae,2002,95:341～349.
[53]尹德明，葛志强，元英进．固定化对红豆杉细胞膜脂肪酸组分和紫杉醇合成的动态影响．高校化学工程学报，2005，19：792～797．
[54]Mori T,Sakarai M.Preparation of conditioned medium to stimulate anthocyanin production using suspension cultures of fragaria ananassa cells.World J Micro Biotechnol,1999,15:635～637.
[55]Carolyn W T,Shuler M L.The effect of inoculum density and conditioned medium on the production of ajmalicine and catharathine from immobilized catharanthus roseus cells.Biotechnol Bioeng,2000,67:61～71.
[56]Ashmore S E.Status report on the development and application of in vitro techniques for the conservation and use of plant genetic resources.1997,Rome:International Plant Genetic Resources Institute.
[57].Bajaj Y P S.Cassava plants from meristem cultures freeze-preserved for three years.Field Crop Res,1983,7:161～167.
[58]Angel F,Barney V E,Tohme Jet al.Stability of cassava plants at the DNA level after retrieval from 10 years of in vitro storage.Euphytica,1996,90:307～313.
[59]Harding K.The methylation status of DNA derived from potato plants recovered from slow growth.Plant Cell Tiss Org Cult,1994,37:31～38.
[60]朱至清．体细胞无性系变异与植物改良．植物学通报，1991，8：1～8．
[61]王正询，刘鸿先，周伯春．香蕉试管苗混倍性变异的研究．遗传学报，2000，27：257～269．
[62]马国斌，王鸣，郑学勤．甜瓜组织培养再生植株中的四倍体变异．园艺学报，1999，26：128～130．
[63]计巧灵，王卫国．新疆紫草的无性繁殖及其再生植株的遗传稳定性初探．植物生理学通讯，2001，37：499～502．
[64]杨金玲，桂耀林，郭仲琛．白木千胚性愈伤组织长期继代培养中的分化能力及染色体稳定性研究．西北植物学报，2000，20：44～47．
[65]杨映根，桂耀林，巍唐等．青木千愈伤组织在继代培养中的分化能力及染色体稳定性研究．植物学报，1994，36：934～939．
[66]Larkin P J,Scowcroft W R.Somaclonal variation:a novel source of variability from cell cultures for plant improvement.Theor Appl Genet,1981,60:197～214.
[67]Lin K H,Lai Y C,Li H C et al.Genetic variation and its relationship to root weight in the sweet potato as revealed by rapd analysis.Scientia Horticulturae,2009,120:2～7.
[68]Jiang H F,Liao B S,Ren X P et al.Comparative assessment of genetic diversity of peanut(arachis hypogaea L.)genotypes with various levels of resistance to bacterial wilt through SSR and AFLP analyses.J Genetic Genom,2007,34:544～554.
[69]Richards E J.DNA methylation and plant development.Trends Genet,1997,13:319～323.
[70]Peraza-Echeverria S,Herrera-Valencia V A,J ames-Kay A.Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive amplification polymorphism(MSAP).Plant Sci,2001,161:359～367.
[71]Chakrabarty D,Yu K W,Paek K Y.Detection ofDNA methylation changes during somatic embryogenesis of siberian ginseng(eleuterococcus senticosus).Plant Sci,2003,165:61～68.
[72]Smykal P,Valledor L,Rodriguez R et al.Assessment of genetic and epigenetic stability in long-term in vitro shoot culture of pea(pisum sativum 1.).Plant Cell Rep,2007,26:1985～1998.
[73]Hao Y J,Deng X X.Stress treatments and DNA methylation affected the somatic embryogenesis of citrus callus.Acta Botanica Sinica,2002,44:673～677.
[74]Yamamoto M P,Onodera Y,Touno S Met al.Synergism between rpbf dof and risbzl bzip activators in the regulation of rice seed expression genes.Plant Physiol,2006,141:1694～1707.
[75]Boyko A,Kovalchuk I.Epigenetic control of plant stress response.Environ Mol Mutagen,2008,49:61～72.
[76]Kuo K C,Mccune R A,Gehrke C W.Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA.Nuleic Acids Res,1980,8:4763～4776.
[77]吴筱丹，王华，楼健．白菜DNA甲基化水平的反相离子对高效液相色谱法研究．理化检验-化学分册，2005，41：5～6．
[78]洪柳，邓秀新．应用MSAP技术对脐橙品种进行DNA甲基化分析．中国农业科学，2005，38：2301～2307．
[79]陈小强，王春国，李秀兰等．大花蕙兰子房授粉前后基因组DNA胞嘧啶甲基化状态的MSAP分析．云南植物研究，2008，30：464～470．
[80]Yu L J,Lan W Z,Qin W M et al.High stable production of taxol in elicited synchronous cultures of taxus chinensis.Process Biochem,2002,38:207～210.
[81]Zhang W,Curtin C,Franeo C.Towards manipulation of post-biosynthetic events in secondary metabolism of plant cell cultures.Enzyme Microb Tech,2002,30:688-696.
[82]Qu J G,Zhang W,Jin M F et al.Effect of homogeneity on cell growth and anthocyanin biosynthesis in suspension cultures of vitis vinifera.Chinese J Biotechnol,2006,22:805～810.
[83]Naill M C,Roberts S C.Cell cycle analysis of taxus suspension cultures at the single cell level as an indicator of culture heterogeneity.Biotechnol Bioeng,2005,90:450～491.
[84]Naill M C,Roberts S C.Flow cytometric identification of paclitaxel-accumulating subpopulations.Biotechnol Prog,2005,21:978～983.
[85]余龙江，蔡永君，兰文智．红豆杉细胞周期时相与紫杉醇诱导合成的关系初探．武汉植物学研究，2001，19：509～512．
[86]曲均革，张卫，金美芳等．细胞均一性对葡萄细胞生长和花青素合成的影响．生物工程学报，2006，22：805～810．
[87]Zimmermann B,Holzgreve W,Wenzel F.Novel real-time quantitative PCR test for trisomy.Clin Chem,2002,48:362～363.
[88].Zabeau M,Vos P.Selective restriction fragment amplification:a general method for DNA fingerprinting.E.P.Application,1993.
[89]Peraza-Echeverria S,Herrera-Valencia V A,James-Kay A.Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive amplification polymorphism(MSAP).Plant Sci,2001,161:359～367.
[90]Momparler R L.Epigenetic therapy of cancer with 5-aza-2'-deoxycytidine(decitabine).Semin Oncol,2005,32:443～451.
[91]Zakharov A F,Egolina N A.Differential spiralization along mammalian mitotic chromosomes.Chromosoma,1972,38:341～365.
[92]杨丽，朱红音．人胃癌细胞肿瘤相关基因的表达与甲基化调控．世界华人消化杂志，2005，13：1493～1498．
[93]Kondo H,Ozaki H,Itoh K.Flowering induced by 5-azacytidine,a DNA demethylating reagent in a short-day plant,perilla frutescens var.crispa.Physiologia Plantarum,2006,127:130～137.
[94]Cheng C Y,Masaaki D,Hirohiko H.Epigenetic regulation of the rice retrotransposon tos17.Mole Genetic Genom,2006,276:378～390.
[95]Burn J E,Bagnall D J,Metzger J D.DNA methylation,vernalization and the initiation of flowering.Proc Natl Acad Sci USA,1993,90:287～291.
[96]李梅兰，曾广文，朱祝军．5-氮杂胞苷促进白菜开花的效应分析．浙江大学学报(农业与生命科学版)，2003，29：287～290．
[97]梅兴国，周爱文．红豆杉细胞同步化的研究．生物技术．2001，11：9～11．
[98].Amoroso M G,Longobardo L,Capparelli R.Real time rt-pcr and flow cytometry to investigate wheat kernel hardness:role of puroindoline genes and proteins.Biotech Lett,2004,26:1731～1737.
[99]Vos P R,Hogers M,Bleeker M et al.AFLP:a new concept for DNA fingerprinting.Nucleic Acids Res,1995,23:4407～4414.
[100]Demeulemeester M A C,Stallen N V,Proft M P D.Degree of DNA methylationin chicory influece of plant age and venalization.Plant Sci,1999,142:101～108.
[101]Deus-Neumann B,Zenk M H.Instability of indole alkaloid production in catharanthus roseus cell suspension cultures.Planta Medica,1984,50:427～431.
[102]许萍，张丕方．关于植物细胞脱分化的研究概况．植物学通报，1996，13：20～24．
[103]．王亚馥，王仑山．枸杞组织培养中过氧化物酶和可溶性蛋白质的变化．实验生物学报，1989，22：1～8．
[104]张洁，段继诚，梁振等．东北红豆杉及其伤愈组织粗提物中紫杉醇的HPLC-ESI-MS/MS分析研究．药学学报，2006，41：863～886．
[105]Demulemeester M A C,Van Stallen N,De Proft M P.Degree ofDNA methylation in chicory(cichorium intybus 1.):influence of plant age and vernalization.Plant Sci,1999,142:101～108.
[106]Lu Y L,Rong T Z,Cao M J.Analysis ofdna methylation in different maize tissues.J Genetic Genom,2008,35:41～48.
[107]Zhao X X,Chai Y,Liu B.Epigenetic inheritance and variation of DNA methylation level and pattern in maize intra-specific hybrids.Plant Sci.2007,172:930～938.
[108]Portis E,Acquadro A,Comino C et al.Analysis of dna methylation during germination of pepper(capsicum annuum 1.)seeds using methylation-sensitive amplification polymorphism(MSAP).Plant Sci,2004,166:169～178.
[109]．陈小强，王春国，李秀兰等．大花蕙兰子房授粉前后基因组DNA胞嘧啶甲基化状态的MSAP分析．云南植物研究，2008，30：464～470．
[110]陆光远，伍晓明，陈碧云等．油菜种子萌发过程中DNA甲基化的MSAP分析．科学通报，2005，50：2750～2756．
[111]Matthes M,Singh R,Sc C et al.Variation in oil palm(elaeis guineensis jacq.)tissue culture-derived regenerants revealed by AFLP with methylation-sensitive enzymes.Theor Appl Genet,2000,102:971～979.
[112]Antequera F,Boyes J,Bird A.High levels of de novo methylation and altered chromatin structure at cpg islands in cell lines.Cell,1990,62:503～514.
[113]Lee H S,Chen Z J.Protein-coding genes are epigenetically regulated in arabidopsis polyploids.Proc Natl Acid Sci USA,1998,98:6753～6758.
[114]Meyer H J,Van Standen J.The in vitro production of an anthocyan in from cell cultures of oxalis linearis.Plant Cell Tissue Organ Culture,1995,40:55～58.
[115]于荣敏，马娜，严春艳．外源激素对何首乌毛状根生长及蒽醌类成分生物合成的影响．生物工程学报，2006，4：619～623．
[116]Fett-Neto A G,Melanson S J,Sakata K.Improved growth and toxol yield in develop ing calli of taxus cuspidate by medium composition odification.Biotechnology,1993,11:731～734.
[117]Ketchum R E B,Gibson D M.Media optimization for maximum biomass production in cell cultures of pacific yew.Plant Cell Tissue Organ Culture,1995,42:185～193.
[118]Salman M N.Establishment of callus and cell suspension cultures from gypsophila paniculata leaf segments and study of the attachment of host cells by erw inia herbicola pv.gyp sophilae.Plant Cell Tissue Organ Culture,2002,69:189～196.
[119].Pastemak T,Miskolczi P,Ayaydin F et al.Exogenous auxin and cytokinin dependent activation of cdks and cell division in leaf protoplast-derived cells of alfalfa.Plant Growth Regulation,2000,32:129～141.
[120]Zhao J,Zhu W H,Hu Q.Enhanced indole alkaloid production in suspension compact callus clusters of catharanthus roseus:impacts of plant growth regulators and sucrose.Plant Growth Regulation,2001,33:33～41.
[121]Alay(?)n-Luaces P,Pagano E,Mroginski Let al.Four glycoside hydrolases are differentially modulated by auxins,cytokinins,abscisic acid and gibberellic acid in apple fruit callus cultures.Plant Cell Tissue Organ Culture,2008,95:257～263.
[122]Tyburski J A,Krzemi(?)ski U,Tretyn A.Exogenous auxin affects ascorbate metabolism in roots of tomato seedlings.Plant Growth Regulation,2008,54:203～215.
[123]Sharma S,Millam S,Hedley Pet al.Molecular regulation of somatic embryogenesis in potato: an auxin led perspective. Plant Molecular Biology. 2008,68: 185-201.
[124] Brown J C L, De Decker M M, Fieldes M A. A comparative analysis of developmental profiles for DNA methylation in 5-azacytidine-induced early-flowering flax lines and their control. Plant Sci, 2008,175: 217-225.
[125] Xiao W, Brown R C,Lemmon B Eet al. Regulation of seed size by hypomethylation of maternal and paternal genomes. Plant Physiol. 2006,142: 1160-1168.
[126] Pavlopoulou A, Kossida S. Plant cytosine-5 DNA methyltransferases: structure, function, and molecular evolution. Genomics, 2007,90: 530-541.
[127] Finnegan E J, Peacock W J, Dennis E S. DNA methylation, a key regulator of plant development and other processes. Curr Opin Genet Dev, 2000,10: 217-223.
[128] Yu L J, Lan W Z, Qin W M et al. Effect of salylic acid on fungal elicitor-induced membrane-liquid peroxdation and taxol production in cell suspension cultures of taxus chinensis. Biotechnol Bioeng, 2001, 37: 477-482.
[129] Yuan Y J, Wei Z J, Wu Z L et al. Improved taxol production in suspension cultures of taxus chinensis var.mairei by in situ extraction combined with precursor feeding and additional carbon source introduction in an airlift loop reactor. Biotechnol Lett, 2001, 23: 1659-1662.
[130] Yanpasisan W, King N, Doran P. Analysis of cell cycle activity and population dynamics in heterogeneous plant cell suspensions using flow cytometry. Biotechnol Bioeng, 1998, 58: 515-528.
[131] Naill M C, Roberts S C. Cell cycle analysis of taxus suspension cultures at the single cell level as an indicator of culture heterogeneity. Biotechnol Bioeng, 2005,90: 450-491.
[132] Muller E, Brown P T, Hartke S. DNA variation in tissue-culture-derived rice plants. Theor Appl Genet, 1990, 80: 673-679.
[133] Devaux P, Kilian A, Kleinhofs A. Anther culture and hordeum bulbosum-derived barley doubled haploids: mutations and methylation. Mol Gen Genet, 1993, 241:674-679.
[134] Tregear J W, Morcillo F, Richaud F et al. Characterization of a defension gene expressed in oil palm inflorescences, induction during tissue culture and possible association with epigenetic somaclonal variation eventa. Exp Bot, 2002, 53: 1387-1396.
[135] Brown P T, Kyozuka J, Sukekiyo Y. Molecular changes in protoplast-derived rice plants. Mol Gen Genet, 1990, 223: 324-328.
[136] Kaeppler S M, Kaeppler H F, Rhee Y. Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol, 2000, 43: 179-188.
[137] Mohapatra K P, Sehgal R N, Sharma R K et al. Evaluation of genetic variation in west himalayan yew and its associates using RAPD and AFLP markers. J Plant Biochem Biotechnol, 2008,17: 161-165.
[138] Corradini P, Edelin C, Bruneau A et al. Architectural and genotypic variation in the clonal shrub taxus canadensis as determined from random amplified polymorphic dna and amplified fragment length polymorphism. Canadian J Bot, 2002, 80:205-219.
[139] Dubreuil M, Sebastiani F, Mayol M et al. Isolation and characterization of polymorphic nuclear microsatellite loci in. Conservation Genetic, 2008, 9:1665-1668.
[140] Pennell R I, Dang D V, Apuya N. Secondary metabolite production via manipulation of genome methylation, 2006.
[2]Dong H D,Zhong J J.Enhanced taxane productivity in bioreactor cultivation of taxus chinensis cells by combining elicitation,sucrose feeding and ethylene incorporation.Enzyme Microb Tech,2002,31:116～121.
[3]Wang Y D,Yuan Y J,Wu J C.Induction studies of methyl jasmonate and salicylic acid on taxane production in suspension cultures of taxus chinensis var.mairei.Biochem Engin J,2004,19:259～326
[4]Croteau R,Ketchum R E B,Long R Met al.Taxol biosynthesis and molecular genetics.Phytochem Reviews,2006,5:75.
[5]Ketchum R E,Gibson D M,Croteau R Bet al.The kinetics of taxoid accumulation in cell suspension cultures of taxus following elicitation with methyl jasmonate.Biotechnol Bioeng,1999,62:97～105.
[6]Yukimune Y,Tabata H,Higashi Yet al.Methyl jasmonate-induced overproduction of paclitaxel and baccatin Ⅲ in taxus cell suspension cultures.Nat.Bioteehnol,1996,14:1129～1132.
[7]Pyo S,Parka H,Song B et al.A large-scale purification of paclitaxel from cell cultures of taxus chinensis,2004,39:1985～1991.
[8]张振学，张海霞，杨庆林．紫杉醇构效关系及其类似物的研究开发进展．天然产物研究与开发，2005，17：810～817．
[9]Zhang W,Curtin C,Franco C.Towards manipulation of post-biosynthetic events in secondary metabolism of plant cell cultures.Enzyme Microb Tech,2002,30:688～696.
[10]Vanisree M,Lee C Y,Lo S F et al.Studies on the production of some important secondary metabolites from medicinal plants by plant tissue cultures.Bot Bull Acad Sin,2004,45:1～22.
[11] Liao Z H, Chen M, Gong Y F et al. A new farnesyl diphosphate synthase gene from taxus media rehder. cloning, characterization and functional complementation. J Iintegr Plant Biol, 2006,48: 692-699.
[12] Jennewein S, Park H,Dejong J Met al. Coexpression in yeast of taxus cytochrome p450 reductase with cytochrome p450 oxygenases involved in taxol biosynthesis. Biotechnol Bioeng, 2005, 89: 588-598.
[13] Walker K D, Klettke K, Akiyama T et al. Cloning, heterologous expression, and characterization of a phenylalanine aminomutase involved in taxol biosynthesis. J Biol Chem, 2004, 279: 53947-53954.
[14] Jennewein S, Long R M, Williams R Met al. Cytochrome p450 taxadiene 5a-hydroxylase, a mechanistically unusual monooxygenase catalyzing the first oxygenation step of taxol biosynthesis. Chem Biol, 2004,11: 379-387.
[15] Chau M D, Jennewein S, Walker K et al. Taxol biosynthesis: molecular cloning and characterization of a cytochrome p450 taxoid 7P-hydroxylase. Chem Biol, 2004, 11:663-672.
[16] Kirschberg T A, Vandeusen C L, Rothbard J B et al. Arginine-based molecular transporters the synthesis and chemical evaluation of releasable taxol-transporter conjugates. Org Lett, 2003, 5: 3459-3462.
[17] Walker K, Croteau R. Molecular cloning of a 10-deacetylbaccatinⅢ-10-o-acetyl transferase cDNA from taxus and functional expression in escherichia coli. Proc Natl Acad Sci USA, 2000,97: 583-587.
[18] Hefner J, Ketchum R E, Croteau R. Cloning and functional expression of a cdna encoding geranylgeranyl diphosphate synthase from taxus canadensis and assessment of the role of this prenyltransferase in cells induced for taxol production. Arch Biochem Biophys, 1998, 360: 62-74.
[19] Tr Ehin C, Ahn I, Perennes C et al. Cloning of upstream sequences responsible for cell cycle regulation of the nicotiana sylvestris cycbl gene. Plant Mol Biol, 1997, 35:667-672.
[20] Hefner J, Rubenstein S M, Ketchum R E et al. Cytochrome p450-catalyzed hydroxylation of taxa-4(5), 11(12)-diene to taxa-4(20), 11(12)-dien-5a-ol: the first oxygenation step in taxol biosynthesis.Chem Biol,1996,3:479～489.
[21]余龙江，蔡永君，兰文智．红豆杉细胞培养生产紫杉醇产量稳定性的探讨．广西植物，2002，22：85～88．
[22]赵春芳，余龙江，李丽琴等．氧化水解法提高红豆杉浸膏中紫杉醇产率的研究．天然产物研究与开发，2006，18：95～99．
[23]Kim B J,Gibson D M,Shuler M L.Effect of subculture and elicitation on instability of taxol production in taxus sp.suspension cultures.Biotechnol Prog,2004,20:1666～1673.
[24]Sierra M I,Van Der Heijden R,Van Der Leer T et al.Stability of alkaloid production in cell suspension cultures of tabernaemontana divaricata during long-term subculture.Plant Cell Tiss Org Cult,1992,28:59～68.
[25]Zhang C H,Fevereiro P S.The effect of heat shock on paclitaxel production in taxus yunnanensis cell suspension cultures:role of abscisic acid-pretreatment.Biotechnol Bioeng,2007,96:506～514.
[26]陈永勤，朱蔚华，吴蕴祺等．不同种类红豆杉愈伤组织的诱导及紫杉醇含量的差异．中草药，2000，31：216～218．
[27]杜亚填，陈建华，许建宇等．植物生长调节剂对南方红豆杉愈伤组织培养和紫杉醇合成的影响．天然产物研究与开发，2006，18：569～576．
[28]盛长忠，王淑芳，王勇等．pH对红豆杉愈伤组织生长、PAL活性和紫杉醇含量的影响．中草药，2001，32：929～931．
[29]Kim J H，Yun J H，Hwang Y S．Production of taxol and relied taxanes in taxus brevifolia call cultures：effect of sugar．Biotechnol Lett，1995，17：101～106．
[30]陈永勤，朱蔚华，吴蕴祺．组培条件对云南红豆杉愈伤组织生长和形成紫杉醇的影响．中国中药杂志，2000，25：269～272．
[31]王艳东，葛志强，马振毅等．外加紫杉醇后南方红豆杉细胞的耐受性及其凋亡的研究．高校化学工程学报，2002，16：421～425．
[32]Larkin P J,Scowcroft W R.Somaclonal variation:a novel source of variability from cell cultures for plant improvement.Theor Appl Genet,1981,60:197～214.
[33]Sun Z Y,Han L,Li Y F.Progress in the study and application of plant smaclonal variation.Acta Agriculture Nuleatae Sinica,2005,6:479～484.
[34]Peredo E L,Angeles Revilla M,Arroyo-Garcia R.Assessment of genetic and epigenetic variation in hop plants regenerated from sequential subcultures of organogenic calli.J Plant Physiol,2006,163:1071～1079.
[35]李晓玲，丛娟，于晓明等．植物体细胞无性系变异研究进展．植物学通报，2008，25：121～128．
[36]Amato F.Cytogenetics of plant cell and tissue culture and their regenerates.Crc Crit Rev Plant Sci,1985,8:72～73.
[37]Johnson S S,Phillips R L,Rines H W.Possible role ofheterochromatin chromosome breakage induced by tissue culture in oats(avena sativa 1.).Genome,1987,29:439～446.
[38]高东迎，郭士伟，李霞等．水稻体细胞无性系变异．植物学通报，2002，19：749～755．
[39]Noro Y,Takano-Shimizu T,Syono K.Genetic variations in rice in vitro cultures at the epsps-rps20 region.Theor Appl Genet,2007,114:705～711.
[40]Brettell R I S,Pallotta M A,Gustafson J P et al.Variation at the nor loci in triticale derived from tissue culture.Theor Appl Genet,1986,71:637～643.
[41]Hirochika H,Sugimoto K,Otsuki Y.Retrotransposons of rice involved in mutations induced by tissue culture.Proc Natl Acad Sci U S A,1996,93:7783～7788.
[42]Phillips R L,Keappler S M,Olhoft P.Genetic instability of plant tissue cultures:breakdown of normal controls.Proc Natl Acad Sci USA,1994,91:5222～5226.
[43]Ng H H,Bird A P.DNA methylation and chromatin modification.Curr Opin Genet Dev,1999,9:158～163.
[44]Curradi M,Izzo A,Badaracco Get al.Molecular mechanisms of gene silencing mediated by DNA methylation.Mol Cell Biol,2002,22:3157～3173.
[45]Long L K,Lin X Y,Zhai J Z et al.Heritable alteration in DNA methylation pattern occurred specifically at mobile elements in rice plants following hydrostatic pressurization.Biochem Biophys Res Commun,2006,340:369～376.
[46].Morrish F M,Hanna W W.The expression and perpetuation of inherent somaclonal variation in regenerants from embryogenic cultures ofpennisetum glaucum(1.)r.br.(pearl millet).Theor Appl Genet,1990,80:409～416.
[47]Peschke V M,Phillips R L.Genetic implication of somaclonal variation in plants.Adv Genet,1992,30:41～75.
[48]刁现民，段胜军，陈振玲等．谷子体细胞无性系变异分析．中国农业科学，1999，32：21～26．
[49]Deus-Neumann B,Zenk M H.Instability of indole alkaloid production in catharanthus roseus cell suspension cultures.Planta Medica,1984,50:427～431.
[50]Kim B J,Gibson D M,Shuler M L.Effect of subculture and elicitation on instability of taxol production in taxus sp.suspension cultures.Biotechnol Prog,2004,20:1666～1673.
[51]Dolezel J,Lucretti S,Novak F J.The influence of 2,4-dichlorophenoxyacetic acid on cell cycle kinetics and sister-chromatid exchange frequency in garlic(allium sativa 1.)meristem cells.Biol Plant,1987,29:253～257.
[52]Rubluoa A,Hernandezb T M,Duvalal K et al.Auxin induced morphogenetic responses in long-term in vitro subcultured mammillaria san-angelensis sanchez-mejorada(cactaceae).Scientia Horticulturae,2002,95:341～349.
[53]尹德明，葛志强，元英进．固定化对红豆杉细胞膜脂肪酸组分和紫杉醇合成的动态影响．高校化学工程学报，2005，19：792～797．
[54]Mori T,Sakarai M.Preparation of conditioned medium to stimulate anthocyanin production using suspension cultures of fragaria ananassa cells.World J Micro Biotechnol,1999,15:635～637.
[55]Carolyn W T,Shuler M L.The effect of inoculum density and conditioned medium on the production of ajmalicine and catharathine from immobilized catharanthus roseus cells.Biotechnol Bioeng,2000,67:61～71.
[56]Ashmore S E.Status report on the development and application of in vitro techniques for the conservation and use of plant genetic resources.1997,Rome:International Plant Genetic Resources Institute.
[57].Bajaj Y P S.Cassava plants from meristem cultures freeze-preserved for three years.Field Crop Res,1983,7:161～167.
[58]Angel F,Barney V E,Tohme Jet al.Stability of cassava plants at the DNA level after retrieval from 10 years of in vitro storage.Euphytica,1996,90:307～313.
[59]Harding K.The methylation status of DNA derived from potato plants recovered from slow growth.Plant Cell Tiss Org Cult,1994,37:31～38.
[60]朱至清．体细胞无性系变异与植物改良．植物学通报，1991，8：1～8．
[61]王正询，刘鸿先，周伯春．香蕉试管苗混倍性变异的研究．遗传学报，2000，27：257～269．
[62]马国斌，王鸣，郑学勤．甜瓜组织培养再生植株中的四倍体变异．园艺学报，1999，26：128～130．
[63]计巧灵，王卫国．新疆紫草的无性繁殖及其再生植株的遗传稳定性初探．植物生理学通讯，2001，37：499～502．
[64]杨金玲，桂耀林，郭仲琛．白木千胚性愈伤组织长期继代培养中的分化能力及染色体稳定性研究．西北植物学报，2000，20：44～47．
[65]杨映根，桂耀林，巍唐等．青木千愈伤组织在继代培养中的分化能力及染色体稳定性研究．植物学报，1994，36：934～939．
[66]Larkin P J,Scowcroft W R.Somaclonal variation:a novel source of variability from cell cultures for plant improvement.Theor Appl Genet,1981,60:197～214.
[67]Lin K H,Lai Y C,Li H C et al.Genetic variation and its relationship to root weight in the sweet potato as revealed by rapd analysis.Scientia Horticulturae,2009,120:2～7.
[68]Jiang H F,Liao B S,Ren X P et al.Comparative assessment of genetic diversity of peanut(arachis hypogaea L.)genotypes with various levels of resistance to bacterial wilt through SSR and AFLP analyses.J Genetic Genom,2007,34:544～554.
[69]Richards E J.DNA methylation and plant development.Trends Genet,1997,13:319～323.
[70]Peraza-Echeverria S,Herrera-Valencia V A,J ames-Kay A.Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive amplification polymorphism(MSAP).Plant Sci,2001,161:359～367.
[71]Chakrabarty D,Yu K W,Paek K Y.Detection ofDNA methylation changes during somatic embryogenesis of siberian ginseng(eleuterococcus senticosus).Plant Sci,2003,165:61～68.
[72]Smykal P,Valledor L,Rodriguez R et al.Assessment of genetic and epigenetic stability in long-term in vitro shoot culture of pea(pisum sativum 1.).Plant Cell Rep,2007,26:1985～1998.
[73]Hao Y J,Deng X X.Stress treatments and DNA methylation affected the somatic embryogenesis of citrus callus.Acta Botanica Sinica,2002,44:673～677.
[74]Yamamoto M P,Onodera Y,Touno S Met al.Synergism between rpbf dof and risbzl bzip activators in the regulation of rice seed expression genes.Plant Physiol,2006,141:1694～1707.
[75]Boyko A,Kovalchuk I.Epigenetic control of plant stress response.Environ Mol Mutagen,2008,49:61～72.
[76]Kuo K C,Mccune R A,Gehrke C W.Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA.Nuleic Acids Res,1980,8:4763～4776.
[77]吴筱丹，王华，楼健．白菜DNA甲基化水平的反相离子对高效液相色谱法研究．理化检验-化学分册，2005，41：5～6．
[78]洪柳，邓秀新．应用MSAP技术对脐橙品种进行DNA甲基化分析．中国农业科学，2005，38：2301～2307．
[79]陈小强，王春国，李秀兰等．大花蕙兰子房授粉前后基因组DNA胞嘧啶甲基化状态的MSAP分析．云南植物研究，2008，30：464～470．
[80]Yu L J,Lan W Z,Qin W M et al.High stable production of taxol in elicited synchronous cultures of taxus chinensis.Process Biochem,2002,38:207～210.
[81]Zhang W,Curtin C,Franeo C.Towards manipulation of post-biosynthetic events in secondary metabolism of plant cell cultures.Enzyme Microb Tech,2002,30:688-696.
[82]Qu J G,Zhang W,Jin M F et al.Effect of homogeneity on cell growth and anthocyanin biosynthesis in suspension cultures of vitis vinifera.Chinese J Biotechnol,2006,22:805～810.
[83]Naill M C,Roberts S C.Cell cycle analysis of taxus suspension cultures at the single cell level as an indicator of culture heterogeneity.Biotechnol Bioeng,2005,90:450～491.
[84]Naill M C,Roberts S C.Flow cytometric identification of paclitaxel-accumulating subpopulations.Biotechnol Prog,2005,21:978～983.
[85]余龙江，蔡永君，兰文智．红豆杉细胞周期时相与紫杉醇诱导合成的关系初探．武汉植物学研究，2001，19：509～512．
[86]曲均革，张卫，金美芳等．细胞均一性对葡萄细胞生长和花青素合成的影响．生物工程学报，2006，22：805～810．
[87]Zimmermann B,Holzgreve W,Wenzel F.Novel real-time quantitative PCR test for trisomy.Clin Chem,2002,48:362～363.
[88].Zabeau M,Vos P.Selective restriction fragment amplification:a general method for DNA fingerprinting.E.P.Application,1993.
[89]Peraza-Echeverria S,Herrera-Valencia V A,James-Kay A.Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive amplification polymorphism(MSAP).Plant Sci,2001,161:359～367.
[90]Momparler R L.Epigenetic therapy of cancer with 5-aza-2'-deoxycytidine(decitabine).Semin Oncol,2005,32:443～451.
[91]Zakharov A F,Egolina N A.Differential spiralization along mammalian mitotic chromosomes.Chromosoma,1972,38:341～365.
[92]杨丽，朱红音．人胃癌细胞肿瘤相关基因的表达与甲基化调控．世界华人消化杂志，2005，13：1493～1498．
[93]Kondo H,Ozaki H,Itoh K.Flowering induced by 5-azacytidine,a DNA demethylating reagent in a short-day plant,perilla frutescens var.crispa.Physiologia Plantarum,2006,127:130～137.
[94]Cheng C Y,Masaaki D,Hirohiko H.Epigenetic regulation of the rice retrotransposon tos17.Mole Genetic Genom,2006,276:378～390.
[95]Burn J E,Bagnall D J,Metzger J D.DNA methylation,vernalization and the initiation of flowering.Proc Natl Acad Sci USA,1993,90:287～291.
[96]李梅兰，曾广文，朱祝军．5-氮杂胞苷促进白菜开花的效应分析．浙江大学学报(农业与生命科学版)，2003，29：287～290．
[97]梅兴国，周爱文．红豆杉细胞同步化的研究．生物技术．2001，11：9～11．
[98].Amoroso M G,Longobardo L,Capparelli R.Real time rt-pcr and flow cytometry to investigate wheat kernel hardness:role of puroindoline genes and proteins.Biotech Lett,2004,26:1731～1737.
[99]Vos P R,Hogers M,Bleeker M et al.AFLP:a new concept for DNA fingerprinting.Nucleic Acids Res,1995,23:4407～4414.
[100]Demeulemeester M A C,Stallen N V,Proft M P D.Degree of DNA methylationin chicory influece of plant age and venalization.Plant Sci,1999,142:101～108.
[101]Deus-Neumann B,Zenk M H.Instability of indole alkaloid production in catharanthus roseus cell suspension cultures.Planta Medica,1984,50:427～431.
[102]许萍，张丕方．关于植物细胞脱分化的研究概况．植物学通报，1996，13：20～24．
[103]．王亚馥，王仑山．枸杞组织培养中过氧化物酶和可溶性蛋白质的变化．实验生物学报，1989，22：1～8．
[104]张洁，段继诚，梁振等．东北红豆杉及其伤愈组织粗提物中紫杉醇的HPLC-ESI-MS/MS分析研究．药学学报，2006，41：863～886．
[105]Demulemeester M A C,Van Stallen N,De Proft M P.Degree ofDNA methylation in chicory(cichorium intybus 1.):influence of plant age and vernalization.Plant Sci,1999,142:101～108.
[106]Lu Y L,Rong T Z,Cao M J.Analysis ofdna methylation in different maize tissues.J Genetic Genom,2008,35:41～48.
[107]Zhao X X,Chai Y,Liu B.Epigenetic inheritance and variation of DNA methylation level and pattern in maize intra-specific hybrids.Plant Sci.2007,172:930～938.
[108]Portis E,Acquadro A,Comino C et al.Analysis of dna methylation during germination of pepper(capsicum annuum 1.)seeds using methylation-sensitive amplification polymorphism(MSAP).Plant Sci,2004,166:169～178.
[109]．陈小强，王春国，李秀兰等．大花蕙兰子房授粉前后基因组DNA胞嘧啶甲基化状态的MSAP分析．云南植物研究，2008，30：464～470．
[110]陆光远，伍晓明，陈碧云等．油菜种子萌发过程中DNA甲基化的MSAP分析．科学通报，2005，50：2750～2756．
[111]Matthes M,Singh R,Sc C et al.Variation in oil palm(elaeis guineensis jacq.)tissue culture-derived regenerants revealed by AFLP with methylation-sensitive enzymes.Theor Appl Genet,2000,102:971～979.
[112]Antequera F,Boyes J,Bird A.High levels of de novo methylation and altered chromatin structure at cpg islands in cell lines.Cell,1990,62:503～514.
[113]Lee H S,Chen Z J.Protein-coding genes are epigenetically regulated in arabidopsis polyploids.Proc Natl Acid Sci USA,1998,98:6753～6758.
[114]Meyer H J,Van Standen J.The in vitro production of an anthocyan in from cell cultures of oxalis linearis.Plant Cell Tissue Organ Culture,1995,40:55～58.
[115]于荣敏，马娜，严春艳．外源激素对何首乌毛状根生长及蒽醌类成分生物合成的影响．生物工程学报，2006，4：619～623．
[116]Fett-Neto A G,Melanson S J,Sakata K.Improved growth and toxol yield in develop ing calli of taxus cuspidate by medium composition odification.Biotechnology,1993,11:731～734.
[117]Ketchum R E B,Gibson D M.Media optimization for maximum biomass production in cell cultures of pacific yew.Plant Cell Tissue Organ Culture,1995,42:185～193.
[118]Salman M N.Establishment of callus and cell suspension cultures from gypsophila paniculata leaf segments and study of the attachment of host cells by erw inia herbicola pv.gyp sophilae.Plant Cell Tissue Organ Culture,2002,69:189～196.
[119].Pastemak T,Miskolczi P,Ayaydin F et al.Exogenous auxin and cytokinin dependent activation of cdks and cell division in leaf protoplast-derived cells of alfalfa.Plant Growth Regulation,2000,32:129～141.
[120]Zhao J,Zhu W H,Hu Q.Enhanced indole alkaloid production in suspension compact callus clusters of catharanthus roseus:impacts of plant growth regulators and sucrose.Plant Growth Regulation,2001,33:33～41.
[121]Alay(?)n-Luaces P,Pagano E,Mroginski Let al.Four glycoside hydrolases are differentially modulated by auxins,cytokinins,abscisic acid and gibberellic acid in apple fruit callus cultures.Plant Cell Tissue Organ Culture,2008,95:257～263.
[122]Tyburski J A,Krzemi(?)ski U,Tretyn A.Exogenous auxin affects ascorbate metabolism in roots of tomato seedlings.Plant Growth Regulation,2008,54:203～215.
[123]Sharma S,Millam S,Hedley Pet al.Molecular regulation of somatic embryogenesis in potato: an auxin led perspective. Plant Molecular Biology. 2008,68: 185-201.
[124] Brown J C L, De Decker M M, Fieldes M A. A comparative analysis of developmental profiles for DNA methylation in 5-azacytidine-induced early-flowering flax lines and their control. Plant Sci, 2008,175: 217-225.
[125] Xiao W, Brown R C,Lemmon B Eet al. Regulation of seed size by hypomethylation of maternal and paternal genomes. Plant Physiol. 2006,142: 1160-1168.
[126] Pavlopoulou A, Kossida S. Plant cytosine-5 DNA methyltransferases: structure, function, and molecular evolution. Genomics, 2007,90: 530-541.
[127] Finnegan E J, Peacock W J, Dennis E S. DNA methylation, a key regulator of plant development and other processes. Curr Opin Genet Dev, 2000,10: 217-223.
[128] Yu L J, Lan W Z, Qin W M et al. Effect of salylic acid on fungal elicitor-induced membrane-liquid peroxdation and taxol production in cell suspension cultures of taxus chinensis. Biotechnol Bioeng, 2001, 37: 477-482.
[129] Yuan Y J, Wei Z J, Wu Z L et al. Improved taxol production in suspension cultures of taxus chinensis var.mairei by in situ extraction combined with precursor feeding and additional carbon source introduction in an airlift loop reactor. Biotechnol Lett, 2001, 23: 1659-1662.
[130] Yanpasisan W, King N, Doran P. Analysis of cell cycle activity and population dynamics in heterogeneous plant cell suspensions using flow cytometry. Biotechnol Bioeng, 1998, 58: 515-528.
[131] Naill M C, Roberts S C. Cell cycle analysis of taxus suspension cultures at the single cell level as an indicator of culture heterogeneity. Biotechnol Bioeng, 2005,90: 450-491.
[132] Muller E, Brown P T, Hartke S. DNA variation in tissue-culture-derived rice plants. Theor Appl Genet, 1990, 80: 673-679.
[133] Devaux P, Kilian A, Kleinhofs A. Anther culture and hordeum bulbosum-derived barley doubled haploids: mutations and methylation. Mol Gen Genet, 1993, 241:674-679.
[134] Tregear J W, Morcillo F, Richaud F et al. Characterization of a defension gene expressed in oil palm inflorescences, induction during tissue culture and possible association with epigenetic somaclonal variation eventa. Exp Bot, 2002, 53: 1387-1396.
[135] Brown P T, Kyozuka J, Sukekiyo Y. Molecular changes in protoplast-derived rice plants. Mol Gen Genet, 1990, 223: 324-328.
[136] Kaeppler S M, Kaeppler H F, Rhee Y. Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol, 2000, 43: 179-188.
[137] Mohapatra K P, Sehgal R N, Sharma R K et al. Evaluation of genetic variation in west himalayan yew and its associates using RAPD and AFLP markers. J Plant Biochem Biotechnol, 2008,17: 161-165.
[138] Corradini P, Edelin C, Bruneau A et al. Architectural and genotypic variation in the clonal shrub taxus canadensis as determined from random amplified polymorphic dna and amplified fragment length polymorphism. Canadian J Bot, 2002, 80:205-219.
[139] Dubreuil M, Sebastiani F, Mayol M et al. Isolation and characterization of polymorphic nuclear microsatellite loci in. Conservation Genetic, 2008, 9:1665-1668.
[140] Pennell R I, Dang D V, Apuya N. Secondary metabolite production via manipulation of genome methylation, 2006.